Blood targeted proteomics: centrifugal filter sample preparation vs dilute-and-shoot

Blood has a complex proteome with a huge span of protein abundances, and we are currently exploring sample preparation strategies addressing this potential challenge. We first evaluated the possibility to simply reduce complexity by fractionating blood samples (prior to targeted proteomics), using combinations of centrifugal filters that are promoted as having differing molar mass (M M ) selectivity. However, systematic fractionation was not possible, as the units had surprisingly unpredictable M M filtration profiles. Hence, labeling implying M M selectivity of centrifugal filters shouldn’t be taken literally. One filter combination (100+50K) however appeared to reduce human serum albumin (HSA) compared with much of the proteome (assessed using gel electrophoresis and staining). However, for our target protein beta-catenin, a “dilute-and-shoot” approach (without any attempt to remove high abundant proteins) was what enabled identification of beta-catenin in blood with nano liquid chromatography mass spectrometry (nano-LC-MS). Thus, minimal sample preparation can be an option in targeted blood proteomics. All blood samples prepared and analyzed (using ultrafast nano-LC-MS) were of considerable complexity, and we document the importance of using external standard spiking and minimum three MS/MS transitions to confirm the presence of target proteins.